Composition, structure and interactions of the Cleavage and Polyadenylation Specificity Factor (CPSF) involved in mammalian mRNA 3' end processing

参与哺乳动物 mRNA 3 末端加工的切割和多聚腺苷酸化特异性因子 (CPSF) 的组成、结构和相互作用

• 批准号: 250983620
• 负责人: Professor Dr. Elmar Wahle
• 金额: --
• 依托单位: Abteilung Allgemeine Biochemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/250983620?language=en
• 关键词: Composition; structure; interactions; Cleavage; Polyadenylation

The ends of eukaryotic mRNAs are generated by a processing reaction in which the mRNA precursor is first shortened by endonucleolytic cleavage and the upstream cleavage fragment is then polyadenylated. Within a large assembly of proteins required for the processing reaction, the heterooligomeric cleavage and polyadenylation specificity factor (CPSF) is considered the central factor: CPSF is involved both in cleavage and polyadenylation, it contains the enzyme catalyzing pre-mRNA cleavage, it recognizes the AAUAAA polyadenylation signal, and it endows poly(A) polymerase with specificity for an AAUAAA-containing substrate RNA. However, neither the composition of CPSF nor its molecular interactions are well understood.We have reconstituted CPSF from six overexpressed subunits.The reconstituted factor is active in polyadenylation. Two of the six subunits are dispensable for this reaction, but are probably essential for pre-mRNA cleavage. As the two other proteins required for polyadenylation, poly(A) polymerase and the nuclear poly(A) binding protein, can be produced in E. coli, a fully reconstituted reaction is now available for mechanistic studies.The composition of the minimal CPSF active in AAUAAA-dependent polyadenylation will be determined with respect to types of subunits and the stoichiometry of their association.The RNA binding specificity of CPSF and the contributions of individual subunits to RNA recognition will be studied. This concerns both the AAUAAA polyadenylation signal and additional sequences possibly recognized by CPSF.The interactions of CPSF subunits with each other and, most importantly, with poly(A) polymerase will be determined. We will ask whether the interaction with poly(A) polymerase is regulated by the length of the poly(A) tail, as postulated in a published model for poly(A) tail length control. We will investigate how CPSF stimulates poly(A) polymerase and whether two other processing factors, cleavage factor I and cleavage stimulation factor, both available in overproduced and purified form, can indeed stimulate polyadenylation in a reconstituted system as has been reported for a less purified system.Symplekin and cleavage factor II will be overexpressed to complete the set of known 3 prime processing factors, and a reconstitution of the cleavage reaction from overproduced and purified proteins will be attempted. If this is not successful, identification of the missing factor(s) will be attempted.Finally, we will collaborate with structural biologists to help elucidate the structure of CPSF by means of cryo-electron microscopy and X-ray crystallography

真核mRNA的末端通过加工反应产生，其中mRNA前体首先通过核酸内切切割缩短，然后上游切割片段被聚腺苷酸化。在加工反应所需的大量蛋白质组装中，异源寡聚切割和多聚腺苷酸化特异性因子（CPSF）被认为是中心因子：CPSF参与切割和多聚腺苷酸化，它含有催化前体mRNA切割的酶，它识别AAUAAA多聚腺苷酸化信号，并且它赋予poly（A）聚合酶对含AAUAAA底物RNA的特异性。然而，CPSF的组成和分子间的相互作用还不清楚，我们用六个过表达的亚基重组了CPSF，重组的因子具有多聚腺苷酸化活性。六个亚基中的两个是该反应的必需亚基，但可能是前体mRNA切割所必需的。 与多聚腺苷酸化所需的另外两种蛋白质--多聚腺苷酸聚合酶和核多聚腺苷酸结合蛋白一样，在大肠杆菌中也能产生。CPSF在AAUAAA依赖的多聚腺苷酸化中的最小活性亚基的组成将根据亚基的类型及其缔合的化学计量来确定，CPSF的RNA结合特异性和单个亚基对RNA识别的贡献将被研究。这涉及AAUAAA多聚腺苷酸化信号和可能被CPSF识别的额外序列。CPSF亚基彼此之间的相互作用，最重要的是，与poly（A）聚合酶的相互作用将被确定。我们将询问与poly（A）聚合酶的相互作用是否受poly（A）尾长度的调节，如在公开的poly（A）尾长度控制模型中所假设的。我们将研究CPSF如何刺激多聚腺苷酸聚合酶，以及两种其他加工因子，裂解因子I和裂解刺激因子，两者都以过度生产和纯化的形式获得，是否确实可以在重构系统中刺激多聚腺苷酸化，如已经报道的用于不太纯化的系统。并尝试从过量生产和纯化的蛋白质重构切割反应。最后，我们将与结构生物学家合作，通过冷冻电子显微镜和X射线晶体学来帮助阐明CPSF的结构